Rapid, clear assessment of apoptosis

Annexin V conjugates are useful for detecting translocated phosphatidylserine (PS), a hallmark of apoptosis. Annexin V conjugates can be used in combination with other dyes, including nucleic acid stains, to accurately assess mixed populations of apoptotic and nonapoptotic cells. We offer annexin V conjugates as stand alone reagents or easy-to-use kits.

Flow cytometric detection of apoptosis with annexin V conjugates

We have collaborated with Nexins Research BV—the original developer of fluorescent phosphatidylserine-binding proteins—to produce annexin V conjugates with superior brightness. These annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine, an indicator of intermediate stages of apoptosis. The difference in fluorescence intensity between apoptotic and nonapoptotic cells stained with our fluorescent annexin V conjugates, as measured by flow cytometry, is typically about 100-fold.

Induce apoptosis in cells using the desired method. A negative control should be prepared by incubating cells in the absence of inducing agent.

Harvest the cells after the incubation period and wash in cold phosphate-buffered saline (PBS).

Pellet the washed cells, discard the supernatants, and resuspend the cells in annexin-binding buffer. Determine the cell density and dilute in annexin-binding buffer to ~1 × 106 cells/mL, preparing a sufficient volume to have 100 μL per assay.

Add 5 μL of the annexin V conjugate to each 100 μL of cell suspension. You may also wish to add an appropriate Invitrogen dead-cell indicator such as propidium iodide, SYTOX Green dye, or SYTOX AADvanced dead cell stain.

Incubate the cells at room temperature for 15 minutes.

After the incubation period, add 400 μL of annexin-binding buffer, mix gently, and keep the samples on ice.

As soon as possible, analyze the stained cells by flow cytometry. Cells labeled with the biotin-X conjugate of annexin V will require the application of a secondary detection agent such as fluorophore-labeled streptavidin. The population should separate into at least two groups: live cells with only a low level of fluorescence, and apoptotic cells that exhibit substantially higher fluorescence intensity. If a dead cell stain is used, dead cells will be labeled with both the dead-cell stain and the annexin V conjugate.